The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)